A Novel Ribosomal-based Method for Studying the Microbial Ecology of Environmental Engineering Systems

A novel ribosomal-based method was applied to examine genetic relationships among microorganisms in the phylogenetic domains of Archaea and Bacteria. Representative unaligned nucleotide sequences of 16s rRNA genes were retrieved from the RDP database. In the domain of Archaea, based on the numbers of certain tri-nucleotides or triplets, the two phyla, Euryarchaeota and Crenarchaeota could be separated. The numbers of certain triplets, especially those containing high guanine and cytosine content such as GGG or CCC, also correlate with the optimum temperatures for growth of the species. In the domain of Bacteria, it was noticed that no single set of triplets or triplet ratios could separate all the phyla at once. However, in certain phylum (Proteobacteria in this study), there were selected triplets whose occurrences could help categorise the sequences into their classes. Although this ribosomal-based method presently requires a substantial amount of data processing and is not standardised in the way that it includes some ad-hoc manual observations and selections of triplets, triplet analysis appears to be a very quick and efficient method in studying the phylogenetic characteristics of microorganisms. These characteristics may be potentially determined in the experiments and then used to for study the microbial ecology of environmental engineering systems. INTRODUCTION The prokaryotes are classified based on the 16s rRNA sequences analysis to reflect their phylogenetic features [1, 2]. Previous studies have observed that the numbers of certain triplets in 16s rRNA sequences are species-specific [3]. Therefore it would be reasonable to assume that this trait of triplets applies to a wider scope, i.e. the number of triplets in 16s rRNA sequences could be phylum-specific, class-specific, etc. It could in turn give rise to a simpler way to identify microorganisms: by means of triplets rather than complete 16s rRNA sequences. Additionally, some genetic information in 16S rRNA gene sequence is lost in conventional 16s rRNA sequence analysis because of the requirements to align sequences and compare unambiguous sequences. This study aims to assess the feasibility of the triplet-based method in investigating the genetic relationships among the microorganisms in Archaea and Bacteria domains. It was also noted in previous studies that the triplets in 16s rRNA sequences reveal some of the phenotypic characteristics [4, 5]. Hence the triplets might reflect some other characteristics of microorganisms as well. In this study, Archaea mocroorganisms with higher optimum temperatures were found to possess a higher GC (guanine and cytosine) content. In addition, the occurrences of selected triplets were observed to correlate with optimum growth temperatures. The domain of Bacteria contains numerous phyla; therefore the range was narrowed down to Proteobacteria in this study. Characteristic triplets were found to help categorise sequences into each of the five classes of Proteobacteria. METHODS Representative unaligned nucleotide sequences of 16s rRNA genes for Archaea and Bacteria were retrieved from the Ribosomal Database Project II home page [6]. Characteristics (e.g. number of occurrences, average, standard deviation, coefficient of variation, etc.) of a total 64 of triplets were obtained by software “DNA Sequence”. The triplet characteristics were compared to similarities and differences among different microorganisms in the domains of Archaea and Bacteria as described in Bergey’s Manual [1-2]. Scatter plots were produced to reveal the results. RESULTS Archaea The Phyla of Euryarchaeota and Crenarchaeota The two domains of Archaea, namely Euryarchaeota and Crenarchaeota, could be separated by a few sets of triplets or selected triplet ratios. One example is shown in the figure below, the CCC/GGU values range from 0.366 to 1.143 for Euryarchaeota, and 1.290 to 2.000 for Crenarchaeota; whereas the GGG/GGU values range from 0.902 to 1.829 for Euryarchaeota and 1.903 to 2.783 for Crenarchaeota (Figure 1.).